L glutamax

NCH93 tumor cells originated from a 64-year-old man suffering from a relapsed anaplastic meningioma (WHO°III) located in the left parieto-occipital region. The tumor sample was first mechanically dissected. Then the resulting cell suspension was cultured in Dulbecco’s minimal Eagle’s medium supplemented with 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA), 2% L-GlutaMAX (Sigma-Aldrich) and 1% Penicillin/Streptomycin (Sigma-Aldrich). Cells were subcultured in a ratio of 1:10 twice a week. Mycoplasma contamination was excluded by 4′,6-diamidino-2-phenylindole staining (Roche). The benign cell line Ben-Men-1 was purchased from DSMZ (Braunschweig, Germany). Cells were grown in the same medium as NCH93, and both were cultured at 37 °C in a humidified environment in a 5% CO₂ atmosphere. Cell lines were authenticated by STR DNA profiling analysis (Leibniz Institute DMSZ, Braunschweig, Germany).

Mitochondrial translation products were labelled using ³⁵S-methionine as previously described (24 ). Fibroblasts were washed twice with methionine/cysteine free DMEM (Life Technologies) supplemented with 1 mM L-glutamax, 96 μg/ml cysteine (Sigma), 1 mM pyruvate and 5% (v/v) dialyzed FBS, and incubated in the same medium for 10 min at 37°C. A total of 100 μg/ml emetine dihydrochloride (Sigma) was then added to inhibit cytosolic translation, before pulse-labelling with 100 μCi [³⁵S]-methionine for 45–60 min. Cells were chased for 10 min at 37°C in regular DMEM with 10% FBS, washed three times with PBS and collected. Labelled cells were lysed in PBS, 0.1% n-dodecyl-D-maltoside (DDM), 1% SDS, 50 units Benzonase (Millipore), 1X protease inhibitor cocktail (Roche). Protein concentration was measured by DC protein assay kit (Biorad) and 20 μg of protein were separated by 12% SDS-PAGE. Gel were fixed and vacuum dried, and exposed to X-ray film for 1 week.

Mouse fibroblasts, 3T6 (ATCC; Manassas, VA, USA; CCL-96), were used for viral production and infection and NIH 3T3 (ATCC; CRL-1658) were used for transfection. Cells were grown at 37 °C in a humidified incubator with a 5% CO₂ atmosphere, using Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 4 mM l-glutamax (Sigma-Aldrich). Plasmid DNA was prepared using a JETSTAR Plasmid Purification Kit (Genomed, Löhne, Germany). Plasmid concentration and purity were determined by nanodrop. Transfections were performed by a nucleofector™ device, using an amaxa-kit V (Lonza, Basel, Switzerland) according to the manufacturer’s instructions.

Peripheral blood mononuclear cells (PBMC) were obtained from peripheral blood density gradient centrifugation as already described. Untouched NK cells were isolated from PBMC using a negative selection human NK cell isolation kit with AutoMACS (both from Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instruction. The purity of CD56⁺ NK cells was >90 % as determined by flow cytometry. NK cells were cultured in “NK cell-medium” Very Low Endotoxin medium VLE RPMI-1640 (Biochrome) supplemented with 10 % FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM l-glutamax (all from Sigma). Purified NK cells were directly used for the experiments. NK cells from 27 healthy donors and from six patients were used in this study.

Mitochondrial translation products were labelled using ³⁵S-methionine³⁷ . Fibroblasts were washed twice with methionine/cysteine-free DMEM (Life Technologies) supplemented with 1 mM L-glutamax, 96 μg/ml cysteine (Sigma), 1 mM pyruvate and 5% (v/v) dialyzed FBS, and incubated in the same medium for 10 min at 37 °C. Then, 100 μg/ml emetine dihydrochloride (Sigma) was added to inhibit cytosolic translation, before pulse labelling with 100 μCi [³⁵S]-methionine for 45–60 min. Cells were chased for 10 min at 37 °C in regular DMEM with 10% FBS, washed three times with PBS and harvested. Labelled cells were lysed in PBS, 0.1% n-dodecyl-D-maltoside (DDM), 1% SDS, 50 U Benzonase (Millipore), 1x protease inhibitor cocktail (Roche). Protein concentration was measured by DC protein assay kit (Biorad) and 20 µg of protein were separated by 12% SDS-PAGE. The gels were then stained with the Coomassie staining solution (50% Methanol (Fisher Scientific), 10% Acetic Acid (Sigma), 0.1% Coomassie Brilliant Blue R250 (Biorad)) to confirm equal loading. Gels were then dried and exposed to phosphor screens (GE Healthcare). The signal was detected by using Typhoon^TM Phosphoimager (GE Healthcare).

Mitochondrial translation products were labeled using ³⁵S‐methionine as described previously (Dalla Rosa et al, 2014) with some modifications. Fibroblasts or HeLa cells were washed twice with methionine‐/cysteine‐free DMEM (Life Technologies) supplemented with 1 mM l‐GlutaMAX, 96 μg/ml cysteine (Sigma), 1 mM pyruvate, and 5% (v/v) dialyzed FBS, and incubated in the same medium for 10 min at 37°C. 100 μg/ml emetine dihydrochloride (Sigma) was then added to inhibit cytosolic translation, before pulse‐labeling with 100 μCi [³⁵S]‐methionine for 45–60 min. Cell were chased for 10 min at 37°C in regular DMEM with 10% FBS, washed three times with PBS, and collected. Labeled cells were lysed in PBS, 0.1% n‐dodecyl‐D‐maltoside (DDM), 1% SDS, 50 units of benzonase (Novagen), and 1:50 (v/v) Roche protease inhibitor cocktail. Protein concentration was measured by Lowry assay, and 20 μg of protein was separated by 12% SDS–PAGE. Gels were dried and exposed to phosphor plates, and the signal was detected by PhosporImager.